Unit 5 - Biotechnology Flashcards
What is Forward Genetics?
studying a phenotype to understand the genotype
What is Reverse Genetics?
studying a genotype to undertand reverse genetics
(if you have a known gene, edit the gene, then see what changes occur in the organism, you can undertstand what the gene does)
GMO
Genetically Modified Organisms
plasmids
extrachromosomal DNA
Selective breeding is a way to make a _____.
GMO
What did Boyer & Cohen do in 1973?
Used RECOMBINANT DNA TEHCNOLOGY to create kanamyacin-resistant bacteria.
Explain the steps used in Recombinant DNA Technology for a bacteria.
- Isolate a plasmid.
- Cut the plasmid open.
- Insert new gene into the open plasmid.
- Rejoin ends of the plasmid with the new gene.
–> recombinant DNA - Insert the upgraded plasmid into bacteria so that the genes can be expressed.
What is Recombinant DNA?
DNA containing genes from different species.
Two Methods of Creating Transgenic Organisms
Gene Knoc Out & Gene Knock In
Gene Knock In
the process of creating a transgenic organism with new characteristcis by inserting a gene
Gene Knock Out
the process of creating a transgenic organism with less characteritics than originally by removing a gene
What process creates an organism with a new characteristic?
Gene Knock In
What process creates an organism that has lost an existing characteristic?
Gene Knock Out
Can both alleles be lost from a genome?
Yes.
What is a Knock Down?
Gene is silenced instead of removed
What is a construct?
a piece of engineered DNA
Homologous Recombination
DNA that is homolgous with the the wild-type DNA is included in the new construct with new DNA so that the construct will incorportate more easily into the organism.
Restriction Endonuclease
hydrolyzes nucleic acids within a sequence, but is restricted to only hydrolyzing certain locations on the seqeunce #findsspecifictargetbeforecutting
Endonuclease
hydrolyzes nucleic acids within a sequence
What 3 big things are needed for cutting & pasting to form recombinant DNA?
Restriction Enzymes
Ligase #Glue
Engineered Nucleases
What acts as “molecular scissors”?
Restriction Enzymes/Restriction Endonucleases
Class I Restriction Enzymes
recognize a sequence, but cuts ~1000 base pairs away
Class III Restriction Enzymes
recognize a sequence, but cuts ~20 base pairs away
What Class of Restriction Enzymes are used in by scientists in Recombinant DNA Technology?
Class II
Class II Restriction Enzymes
recognize a sequence of DNA and cuts right there #precise
Which class of restriction enzymes is the most precise?
Class II
HaeIII (Pronunciation & Name Breakdown)
Hey - 3
Hae = Haemophilus aegyptus
3 = this was the 3rd restriction endonuclease reconginzied from H. aegyptus
HindIII (Name Breakdown)
H=Haemophilus
in = influenzae
d = strain of H. influenzae
3 = 3rd endonuclease discovered in that strain of bacteria
What type of cut does HindIII make?
Cohesive Cut
What type of cut does HaeIII make?
Blunt Cut
Cohesive Cut
staggered cut
(leaves behind single-strandeded overhangs #stickyends)
*effective in attaching molecules with similar sticky ends.
Blunt Cut
straight cut through both strands of DNA
What do bacteria use restrcition endonuclease activity for?
Restriction endonucleases allow bacteria to protect themselves against viral DNA by cutting up the viral DNA.
What determines how frequently a restriction enzyme cuts?
How long or short the restriction sequence is.
The shorter the sqeuence, the more frequently it cuts. The longer the sequence, the more restricted it is and the less frequently it cuts.
Ligase
seals the sugar-phosphate backbone of DNA by creating phosphodiester bonds #glue
Can temperature affect restriction enzymes?
Yes
Because enzymes are proteins and can be denatured.
At what temp do most restriction enzymes work best?
37 C
Do restriction enzymes need cofactors?
Sometimes - depending on which enzyme.
5 Factors Affecting the Activity of Restriction Enzymes:
- temp (~37)
- presence of cofactors (i.e. Mg)
- conc. of ions
- pH (~7-8)
- methylation of DNA
Can nucleases be programmed to cut specific sequences in genome editing?
Yes
i.e. Meganucleases, ZFN, TALEN, Cas9
Which nuclease is the easiest to engineer?
Cas9
ZFN
Zinc Finger Nucleases
TALEN
Transcription Activator Like-Effector Nucleases
How does Cas9 differ from other nuleases in regards to how it knows where to cut DNA?
Most nucleases are a protein which binds to a sequence of DNA.
Cas9 is a protein that will bind to DNA, but it also contains a sequence of guide RNA which base pairs with the DNA it will cut. #specific
What are 5 Ways DNA can be Manipulated?
- Cutting & Pasting
- Transferring & Storing
- Sorting & Sequencing
- Amplifying (PCR)
- GWAS
GWAS
Genome Wide Association Studies
PCR
Polymerase Chain Reaction
What is a Cloning Vector?
The DNA which will have foreign DNA inserted.
(It will become a Recombinant DNA molecule after)
Cloning Site
seqeunces of DNA in a plasmid recognized by endonucleases where it will be cut and opened
The sitcky ends can then be joined with the sticky ends of a foreign DNA that has been cut by the same nuclease.
Cloning Site AKA
AKA Multiple Cloning Site/Poly Linker
Explain How Two Pieces of DNA are Joined Together
Sticky ends of two DNA fragments base pair and are held together with hydroden bonds between the bases.
Ligase comes along and seals the sugar-phosphate backbone with phosphodiester bonds.
Good Cloning Vectors Have:
- Only 1 Restriction Site/Cloning Site
- Origin of Replication (so cloning can actually occur)
- Selectable Marker
Selectable Marker
Help one ID which bacterial cells have taken up the vector with the recombinant DNA.
The selctable marker is a gene helping the bacteria to survive certain conditions such as exposure to an antibiotic – this allows the bacteria with the vector be ID’d.
Isoschizmer
An endonucleases which recognizes and cuts at the same sequence as another endonuclease.
Types of Cloning Vectors
plasmids, phages, cosmids, artificial chromosomes
Cosmids
intermediate b/w plasmids and phages
oriC
Origin of replication site for a Chromosome
Two Types of Artificial Chromosomes
Yeast and Bacterial Artifical Chromosomes
Two Ways an Engineered Vector with Recombinant DNA can get into a cell to be Cloned:
- Phage/Viral Infection
- Transforming Bacteria
What is Transformation of Bacteria?
Bacteria take up naked DNA from their enviroment into their cell then incorporate it into their DNA.
Competent Cell
bacteria which are more likely to take up DNA from their environment for transformation
Explain Blue-White Screening
The vector plasmid has the cloning site, origin, and selective marker (ampicillin resistance) PLUS a lacZ gene.
The foreign DNA is inserted into the lacZ gene at the restriction site and causes the plasmid to be lacZ- and NOT produce beta-galactosidase.
The bacteria is then plated on a medium w/ ampicillin and X-gal.
Bacteria with no plasmid (and there for no ampicillin resisntance) DIE.
Bacteria with nonrecombinant DNA (no foreign DNA) turn BLUE because they still make beta-galactosidase and the X-gal makes the beta-galactosidase blue.
Bacteria with the recombinant DNA (foreign DNA) will look WHITE because the lacZ gene is broken up and cannot produce the beta-galactosidase and therefore not use the x-gal to turn blue
What does lacZ gene code for?
Beta-galactosidase
In Blue-White Plating, what color are bacteria with nonrecombinant DNA?
Blue
(because the lacZ gene is still in one piece and can use the X-gal which turns the beta-galactosidase produced by the lacZ gene blue)
In Blue-White Plating, what color are bacteria with recombinant DNA?
White
(because the foreign DNA has been inserted into the lacZ gene meaning lacZ gene is broken and can no longer produce beta-galactosidase. Because no beta-galactosidase is produced the X-gal is not used to turn the colony blue and stays white instead)
What are the 2 types of Vectors?
- Cloning Vectors
- Expression Vectors
What are 2 sources of DNA inserts?
- Genomic Fragments
- cDNA
What is the biggest reason for using Cloning Vectors over Expression Vectors?
Cloning Vectors are good to AMPLIFY the foreign DNA (rather than express the gene)
#replicate
What is the benefit of using Expression Vectors over Cloning Vectors?
Expression Vectors have promoters, regulatory sequences, etc to express the new gene.
What bacteria fuctions as a good Expression Vector?
E. coli
cDNA
complimentary DNA
DNA sequences were being transcribed into mRNA in the parent cell.
The mRNA is harvested. The mRNA is convered back into DNA and then the DNA is digested and incorporated into the vectors.
What is the difference between genomic fragment and cDNA libraries?
Libraries made up of genomic fragments contain ALL the bits and pieces of a genome.
cDNA libraries contain only the genes in DNA that were turned on/expressed and actively being transcribed in the parent cell.
What process converts mRNA into cDNA?
reverse transcription
What enzyme converts mRNA into cDNA?
viral reverse transcriptase
then DNA polymerase forms the 2nd strand of DNA from the 1st strand which was created using the viral reverse transcriptase
What sequences would you find in a genomic library that would NOT be present in a cDNA library?
You would find sequences of DNA that are not expressed.
i.e. intron, promoter, telomere, centromere, tRNA sequences, etc.
What is the advantage of a cDNA library?
You can narrow down which genes are being expressed vs not expressed.
Electroporation
Brute force that uses electric forces to force bacterial atrifical chromosome cloning vectors into a cell because the vectors are too large to naturally enter on their own
Gel Electrophoresis separates molecules by _____.
size
Gel Electrophoreis
An electric force pushes molecules through a porous gel polymer (i.e. SDS-PAGE). Smaller molecules will move further and larger molecules will move less.
Negative Electrode is located near the wells where the molecules are inserted and the Positive Electrode is located at the end where molecules are moving toward.
SDS-PAGE
sodium dodecyl-sulfate polyacrylamide gel electrophoresis
Purpose of using SDS (sodium dodecyl-sulfate) in Gel Electrophoresis
makes proteins more linear and more negatively charged so they can move through the gel easier
Classic Method of DNA Sequencing
Sanger (dideoxy) Method
Sanger (dideoxy) Method of DNA Sequencing
Make copies of DNA with d and dd NTPs. “Replication” of the DNA will continue if a dNTP is inserted, but will halt if the ddNTP is inserted becasue it lacks a 3’ OH group.
Some copies will go further than others. You can compare sequences of all the chunks and overlap congruent areas to eventually figure the entire sequence.
dNTP
deoxyribonucleotide triphosphates
ddNTPS
dideoxyribonucleotide triphosphates
No 3’ OH group –> replication stops because the next nucleotide in the DNA sequence cannot bond to it.
hydroxyl group
-OH
How are the different fragments produced in the Sanger (dideoxy) Method separated?
gel electrophoresis
If reading a gel electrophoresis to determine a DNA sequence, the _____ strand of nucleotides would be found at end closest to the (+)Electrode and be representative of the __’ end.
shortest
5’ end of the complimentary strand
Cycle Sequencing
Modern Version of the Sanger Method of DNA Sequencing – uses florescently labeled nucleotides which can be detected by a machine.
Which Type of DNA Sequencing allows multiple strands of DNA to be sequenced simultaneously?
Next Generation Sequencing (NGS)
NGS
Next Generation Sequencing
What is the Original Method of DNA Sequencing?
The Sanger Method
What is the more modern version of the Sanger Method of DNA Sequencing?
Cycle Sequencing
Which method of DNA Sequencing is the fastest and cheapest?
Next Generation Sequencing
What enzyme/molecule in Next Generation Sequencing is responsible for producing the light that is measured?
luciferase
Explain the Basics of How NGS Works
- DNA is broken into fragments.
- The fragments are made single stranded.
- Each fragment is attached to a bead and amplified
- The bead with fragmented ssDNA is forced into a well where DNA replication can occur
- A solution with 1 type of nucleotide at a time is run over the wells. As synthesis occurs 1 nucleotide at a time, luciferase is activated by P released and emits light which can be measured.
How does luciferase emit light?
As dNTPs are incorporated into DNA, a P is released. The P is then added to ADP to form ATP which is then used to power luciferase which emits light.
PCR
Polymerase Chain Reaction
What does PCR allow?
PCR allows one to quickly amplify (replicate) short sequences of DNA
What is the recipe for PCR?
- template DNA
- primers
- nucleotides (dNTPs)
- PCR buffer
- Taq DNA polymerase
- water
Steps Used to Amplify DNA in PCR
- Denaturation (dsDNA –> ssDNA)
- Annealing (adding primers to ssDNA)
- Elongation
- Repeat
How is each step in PCR controlled?
by changing the temp
How many cycles of PCR does it take to get the target sequence?
3
hybridization of nucleic acids
complementary sequencing of ssDNA or RNA figure out how to align and interact with hydrogen bonds
Microsatellites AKA
Short Tandem Repeats (STRs)
Microsatellites
short tandem repeats of nucleotides. Can vary in number of nucleotides in the sequences and the number of repeats
CODIS
Combined DNA Index System
What system is used by FBI for DNA fingerprinting?
CODIS
How many microsatellites are used in CODIS?
13
